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Scientific Reports May 2018The CRISPR/Cas9 system provides a novel and promising tool for editing the HIV-1 proviral genome. We designed RNA-guided CRISPR/Cas9 targeting the HIV-1 regulatory genes...
The CRISPR/Cas9 system provides a novel and promising tool for editing the HIV-1 proviral genome. We designed RNA-guided CRISPR/Cas9 targeting the HIV-1 regulatory genes tat and rev with guide RNAs (gRNA) selected from each gene based on CRISPR specificity and sequence conservation across six major HIV-1 subtypes. Each gRNA was cloned into lentiCRISPRv2 before co-transfection to create a lentiviral vector and transduction into target cells. CRISPR/Cas9 transduction into 293 T and HeLa cells stably expressing Tat and Rev proteins successfully abolished the expression of each protein relative to that in non-transduced and gRNA-absent vector-transduced cells. Tat functional assays showed significantly reduced HIV-1 promoter-driven luciferase expression after tat-CRISPR transduction, while Rev functional assays revealed abolished gp120 expression after rev-CRISPR transduction. The target gene was mutated at the Cas9 cleavage site with high frequency and various indel mutations. Conversely, no mutations were detected at off-target sites and Cas9 expression had no effect on cell viability. CRISPR/Cas9 was further tested in persistently and latently HIV-1-infected T-cell lines, in which p24 levels were significantly suppressed even after cytokine reactivation, and multiplexing all six gRNAs further increased efficiency. Thus, the CRISPR/Cas9 system targeting HIV-1 regulatory genes may serve as a favorable means to achieve functional cures.
Topics: CRISPR-Cas Systems; Cell Line; Genes, rev; Genes, tat; Genetic Therapy; HIV-1; Humans; T-Lymphocytes; Virus Replication
PubMed: 29773895
DOI: 10.1038/s41598-018-26190-1 -
Journal of Virology Dec 1991Transcriptional regulatory mechanisms found in lentiviruses employ RNA enhancer elements called trans-activation responsive (TAR) elements. These nascent RNA stem-loops... (Comparative Study)
Comparative Study
Transcriptional regulatory mechanisms found in lentiviruses employ RNA enhancer elements called trans-activation responsive (TAR) elements. These nascent RNA stem-loops are cis-acting targets of virally encoded Tat effectors. Interactions between Tat and TAR increase the processivity of transcription complexes and lead to efficient copying of viral genomes. To study essential elements of this trans activation, peptide motifs from Tats of two distantly related lentiviruses, equine infectious anemia virus (EIAV) and human immunodeficiency virus type 1 (HIV-1), were fused to the coat protein of bacteriophage R17 and tested on the long terminal repeat of EIAV, where TAR was replaced by the R17 operator, the target of the coat protein. This independent RNA-tethering mechanism mapped activation domains of Tats from HIV-1 and EIAV to 47 and 15 amino acids and RNA-binding domains to 10 and 26 amino acids, respectively. Thus, a minimal lentivirus Tat consists of 25 amino acids, of which 15 modify viral transcription and 10 bind to the target RNA stem-loop.
Topics: Amino Acid Sequence; Base Sequence; Binding Sites; Capsid; Enhancer Elements, Genetic; Gene Expression Regulation, Viral; Genes, tat; HIV-1; Infectious Anemia Virus, Equine; Lentivirus; Molecular Sequence Data; Plasmids; RNA, Viral; Repetitive Sequences, Nucleic Acid; Sequence Homology, Nucleic Acid; Transcription, Genetic; Transcriptional Activation
PubMed: 1658392
DOI: 10.1128/JVI.65.12.7012-7015.1991 -
Experimental Hematology Nov 2001Molecular imaging can be broadly defined as the in vivo characterization and measurement of biological processes at the cellular and molecular level. In contrast to... (Review)
Review
Molecular imaging can be broadly defined as the in vivo characterization and measurement of biological processes at the cellular and molecular level. In contrast to commonly used clinical imaging, it sets forth to probe the molecular abnormalities that are the basis of disease, rather than imaging the end effects of these molecular alterations. Development of new imaging technologies requires a multidisciplinary collaboration between biologists, chemists, physicists, and imaging scientists to create novel agents, signal amplification strategies, and imaging techniques that successfully address these questions. In this article we attempt to present some of the recent developments and show how molecular imaging can be used, at least experimentally, to assess specific molecular targets for gene- and cell-based therapies. In particular, we place emphasis on the development and use of experimental small-animal models, which are particularly inclined toward this approach, primarily in combination with magnetic resonance (MR), radionuclide, and optical imaging. In the future, specific imaging of disease targets will allow earlier detection and characterization of disease, as well as earlier and direct molecular assessment of treatment efficacy.
Topics: Animals; Cell Movement; Cell- and Tissue-Based Therapy; Diagnostic Imaging; Ferric Compounds; Forecasting; Gene Expression; Gene Products, tat; Genes, Reporter; Genetic Therapy; Humans; Image Processing, Computer-Assisted; Luminescent Measurements; Magnetic Resonance Imaging; Mice; Mice, Transgenic; Microscopy; Models, Animal; Protein Sorting Signals; Sensitivity and Specificity; Staining and Labeling; Tomography; Tomography, Emission-Computed; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; Ultrasonics
PubMed: 11698119
DOI: 10.1016/s0301-472x(01)00739-1 -
Free Radical Biology & Medicine Nov 2011Chronic human immunodeficiency virus infection is associated with higher incidence of pulmonary complications including hypertension, vasculopathy, lymphocytic...
Chronic human immunodeficiency virus infection is associated with higher incidence of pulmonary complications including hypertension, vasculopathy, lymphocytic alveolitis, and interstitial pneumonitis not attributed to either opportunistic infections or presence of the virus. The Tat (transactivator of transcription) protein, a required transactivator for expression of full-length viral genes, is pleiotropic and influences expression of cellular inflammatory genes. Tat-dependent transactivation of cellular genes requires specific mediators, including NF-κB, widely recognized as sensitive to changes in cellular oxidant burden. We hypothesized that overproduction of Tat leads to increased oxidant burden and to alterations in basal inflammatory status as measured by NF-κB activation. We engineered transgenic mouse lines that express Tat (86-amino-acid isoform) in the lung under the control of the surfactant protein C promoter. Tat-transgenic mice exhibit increased pulmonary cellular infiltration, increased nitrotyrosine and carbonyl protein modifications, and increased levels of NF-κB, MnSOD, and thioredoxin-interacting protein. These data indicate that Tat increases oxidant burden and resets the threshold for inflammation, which may increase susceptibility to secondary injuries.
Topics: Animals; Female; Gene Products, tat; Gene Transfer Techniques; HIV-1; Inflammation; Lung; Male; Mice; Mice, Transgenic; NF-kappa B; Oxidative Stress
PubMed: 21855628
DOI: 10.1016/j.freeradbiomed.2011.07.023 -
Retrovirology Apr 2014We have shown that HIV-1 Tat interaction with MAP2K3, MAP2K6, and IRF7 promoters is key to IFN-stimulated genes (ISG) activation in immature dendritic cells and...
BACKGROUND
We have shown that HIV-1 Tat interaction with MAP2K3, MAP2K6, and IRF7 promoters is key to IFN-stimulated genes (ISG) activation in immature dendritic cells and macrophages.
RESULTS
We evaluated how Tat alleles and mutants differ in cellular gene modulation of immature dendritic cells and monocyte-derived macrophages and what similarities this modulation has with that induced by interferons. The tested alleles and mutants modulated to different degrees ISG, without concomitant induction of interferons. The first exon TatSF21-72 and the minimal transactivator TatSF21-58, all modulated genes to a significantly greater extent than full-length wild type, two-exon Tat, indicating that Tat second exon is critical in reducing the innate response triggered by HIV-1 in these cells. Mutants with reduced LTR transactivation had a substantially reduced effect on host gene expression modulation than wild type TatSF2. However, the more potent LTR transactivator TatSF2A58T modulated ISG expression to a lower degree compared to TatSF2. A cellular gene modulation similar to that induced by Tat and Tat mutants in immature dendritic cells could be observed in monocyte-derived macrophages, with the most significant pathways affected by Tat being the same in both cell types. Tat expression in cells deleted of the type I IFN locus or receptor resulted in a gene modulation pattern similar to that induced in primary immature dendritic cells and monocyte-derived macrophages, excluding the involvement of type I IFNs in Tat-mediated gene modulation. ISG activation depends on Tat interaction with MAP2K3, MAP2K6, and IRF7 promoters and a single exon Tat protein more strongly modulated the luciferase activity mediated by MAP2K3, MAP2K6, and IRF7 promoter sequences located 5' of the RNA start site than the wild type two-exon Tat, while a cysteine and lysine Tat mutants, reduced in LTR transactivation, had negligible effects on these promoters. Chemical inhibition of CDK9 or Sp1 decreased Tat activation of MAP2K3-, MAP2K6-, and IRF7-mediated luciferase transcription.
CONCLUSIONS
Taken together, these data indicate that the second exon of Tat is critical to the containment of the innate response stimulated by Tat in antigen presenting cells and support a role for Tat in stimulating cellular transcription via its interaction with transcription factors present at promoters.
Topics: Alleles; Amino Acid Sequence; Antigen-Presenting Cells; Dendritic Cells; Exons; Gene Expression Regulation, Viral; HIV-1; Humans; Interferon Regulatory Factor-7; Interferon Type I; MAP Kinase Kinase 3; MAP Kinase Kinase 6; Macrophages; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Transcriptional Activation; tat Gene Products, Human Immunodeficiency Virus
PubMed: 24742347
DOI: 10.1186/1742-4690-11-30 -
Annual Review of Virology Sep 2017Viruses are completely dependent upon cellular machinery to support replication and have therefore developed strategies to co-opt cellular processes to optimize... (Review)
Review
Viruses are completely dependent upon cellular machinery to support replication and have therefore developed strategies to co-opt cellular processes to optimize infection and counter host immune defenses. Many viruses, including human immunodeficiency virus type 1 (HIV-1), encode a relatively small number of genes. Viruses with limited genetic content often encode multifunctional proteins that function at multiple stages of the viral replication cycle. In this review, we discuss the functions of HIV-1 regulatory (Tat and Rev) and accessory (Vif, Vpr, Vpu, and Nef) proteins. Each of these proteins has a highly conserved primary activity; however, numerous additional activities have been attributed to these viral proteins. We explore the possibility that HIV-1 proteins leverage their multifunctional nature to alter host transcriptional networks to elicit a diverse set of cellular responses. Although these transcriptional effects appear to benefit the virus, it is not yet clear whether they are strongly selected for during viral evolution or are a ripple effect from the primary function. As our detailed knowledge of these viral proteins improves, we will undoubtedly uncover how the multifunctional nature of these HIV-1 regulatory and accessory proteins, and in particular their transcriptional functions, work to drive viral pathogenesis.
Topics: Genes, rev; Genes, tat; HIV-1; Host-Pathogen Interactions; Human Immunodeficiency Virus Proteins; Humans; Transcription, Genetic; Viral Regulatory and Accessory Proteins; Virus Replication; nef Gene Products, Human Immunodeficiency Virus; vif Gene Products, Human Immunodeficiency Virus; vpr Gene Products, Human Immunodeficiency Virus
PubMed: 28961413
DOI: 10.1146/annurev-virology-101416-041654 -
International Journal of Molecular... Mar 2024Viruses provide vital insights into gene expression control. Viral transactivators, with other viral and cellular proteins, regulate expression of self, other viruses,...
Viruses provide vital insights into gene expression control. Viral transactivators, with other viral and cellular proteins, regulate expression of self, other viruses, and host genes with profound effects on infected cells, underlying inflammation, control of immune responses, and pathogenesis. The multifunctional Tat proteins of lentiviruses (HIV-1, HIV-2, and SIV) transactivate gene expression by recruiting host proteins and binding to transacting responsive regions (TARs) in viral and host RNAs. SARS-CoV-2 nucleocapsid participates in early viral transcription, recruits similar cellular proteins, and shares intracellular, surface, and extracellular distribution with Tat. SARS-CoV-2 nucleocapsid interacting with the replication-transcription complex might, therefore, transactivate viral and cellular RNAs in the transcription and reactivation of self and other viruses, acute and chronic pathogenesis, immune evasion, and viral evolution. Here, we show, by using primary and secondary structural comparisons, that the leaders of SARS-CoV-2 and other coronaviruses contain TAR-like sequences in stem-loops 2 and 3. The coronaviral nucleocapsid C-terminal domains harbor a region of similarity to TAR-binding regions of lentiviral Tat proteins, and coronaviral nonstructural protein 12 has a cysteine-rich metal binding, dimerization domain, as do lentiviral Tat proteins. Although SARS-CoV-1 nucleocapsid transactivated gene expression in a replicon-based study, further experimental evidence for coronaviral transactivation and its possible implications is warranted.
Topics: Humans; HIV-1; SARS-CoV-2; Transcriptional Activation; HIV Long Terminal Repeat; COVID-19; Gene Products, tat; Lentivirus; Gene Expression; tat Gene Products, Human Immunodeficiency Virus; RNA, Viral
PubMed: 38542351
DOI: 10.3390/ijms25063378 -
Journal of Bacteriology Nov 2006The Tat system allows the translocation of folded and often cofactor-containing proteins across biological membranes. Here, we show by an interspecies transfer of a... (Comparative Study)
Comparative Study
The Tat system allows the translocation of folded and often cofactor-containing proteins across biological membranes. Here, we show by an interspecies transfer of a complete Tat translocon that Tat systems are largely, but not fully, interchangeable even between different classes of proteobacteria. The Tat apparatus from the alpha-proteobacterium Rhodobacter capsulatus was transferred to a Tat-deficient Escherichia coli strain, which is a gamma-proteobacterium. Similar to that of E. coli, the R. capsulatus Tat system consists of three components, rc-TatA, rc-TatB, and rc-TatC. A fourth gene (rc-tatF) is present in the rc-tatABCF operon which has no apparent relevance for translocation. The translational starts of rc-tatC and rc-tatF overlap in four nucleotides (ATGA) with the preceding tat genes, pointing to efficient translational coupling of rc-tatB, rc-tatC, and rc-tatF. We show by a variety of physiological and biochemical assays that the R. capsulatus Tat system functionally targets the E. coli Tat substrates TorA, AmiA, AmiC, and formate dehydrogenase. Even a Tat substrate from a third organism is accepted, demonstrating that usually Tat systems and Tat substrates from different proteobacteria are compatible with each other. Only one exceptional Tat substrate of E. coli, a membrane-anchored dimethyl sulfoxide (DMSO) reductase, was not targeted by the R. capsulatus Tat system, resulting in a DMSO respiration deficiency. Although the general features of Tat substrates and translocons are similar between species, the data indicate that details in the targeting pathways can vary considerably.
Topics: Bacterial Proteins; Escherichia coli; Escherichia coli Proteins; Genetic Complementation Test; Iron-Sulfur Proteins; Membrane Transport Proteins; Oxidoreductases; Rhodobacter capsulatus; Substrate Specificity; Transfection
PubMed: 16980457
DOI: 10.1128/JB.01139-06 -
PloS One 2017Despite the success of cART, greater than 50% of HIV infected people develop cognitive and motor deficits termed HIV-associated neurocognitive disorders (HAND)....
Despite the success of cART, greater than 50% of HIV infected people develop cognitive and motor deficits termed HIV-associated neurocognitive disorders (HAND). Macrophages are the major cell type infected in the CNS. Unlike for T cells, the virus does not kill macrophages and these long-lived cells may become HIV reservoirs in the brain. They produce cytokines/chemokines and viral proteins that promote inflammation and neuronal damage, playing a key role in HIV neuropathogenesis. HIV Tat is the transactivator of transcription that is essential for replication and transcriptional regulation of the virus and is the first protein to be produced after HIV infection. Even with successful cART, Tat is produced by infected cells. In this study we examined the role of the HIV Tat protein in the regulation of gene expression in human macrophages. Using THP-1 cells, a human monocyte/macrophage cell line, and their infection with lentivirus, we generated stable cell lines that express Tat-Flag. We performed ChIP-seq analysis of these cells and found 66 association sites of Tat in promoter or coding regions. Among these are C5, CRLF2/TSLPR, BDNF, and APBA1/Mint1, genes associated with inflammation/damage. We confirmed the association of Tat with these sequences by ChIP assay and expression of these genes in our THP-1 cell lines by qRT-PCR. We found that HIV Tat increased expression of C5, APBA1, and BDNF, and decreased CRLF2. The K50A Tat-mutation dysregulated expression of these genes without affecting the binding of the Tat complex to their gene sequences. Our data suggest that HIV Tat, produced by macrophage HIV reservoirs in the brain despite successful cART, contributes to neuropathogenesis in HIV-infected people.
Topics: AIDS Dementia Complex; Adaptor Proteins, Signal Transducing; Amino Acid Substitution; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line; Complement C5; Gene Expression Regulation; Humans; Macrophages; Nerve Tissue Proteins; Receptors, Cytokine; tat Gene Products, Human Immunodeficiency Virus
PubMed: 28640909
DOI: 10.1371/journal.pone.0179882 -
Molecular Pharmacology May 2008Increased deposition of amyloid beta (Abeta) is characteristic for normal aging and human immunodeficiency virus-1 (HIV-1)-associated alterations of the central nervous...
Increased deposition of amyloid beta (Abeta) is characteristic for normal aging and human immunodeficiency virus-1 (HIV-1)-associated alterations of the central nervous system. In addition, both Abeta and HIV-1 are known to induce cellular oxidative stress and disruption of the blood-brain barrier (BBB). Therefore, we hypothesize that Abeta and HIV-1 protein Tat can potentiate their proinflammatory effects at the brain endothelium level. To address this hypothesis, we studied promoter activity of three proinflammatory genes in an in vitro BBB model of human brain microvascular endothelial cells (HBMEC) cocultured with a human astrocyte cell line producing Tat (SVGA-Tat cells) and exposed to Abeta. Treatment of HBMEC with Abeta(1-40) in the presence of SVGA-Tat cells resulted in a significant up-regulation of E-selectin, CC chemokine ligand-2, and interleukin-6 promoter activities and protein levels compared with the individual effects of Abeta or Tat. In addition, Abeta markedly amplified E-selectin promoter activity in HBMEC cocultured with HIV-1-infected Jurkat T cells. Simvastatin, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, effectively blocked proinflammatory reactions induced by Abeta in cocultures with SVGA-Tat cells or with HIV-1-infected Jurkat cells. The present study indicates that a combined exposure to Abeta and Tat or HIV-1 can synergistically potentiate the expression of inflammatory genes in brain endothelial cells. In addition, simvastatin may provide a beneficial influence by reducing these effects at the BBB level.
Topics: Amyloid beta-Peptides; Anticholesteremic Agents; Astrocytes; Brain; Cell Line, Transformed; Cytoprotection; Endothelial Cells; Gene Expression Regulation; HIV-1; Humans; Inflammation; Inflammation Mediators; Jurkat Cells; Peptide Fragments; Promoter Regions, Genetic; Selectins; Simvastatin; Transcriptional Activation; tat Gene Products, Human Immunodeficiency Virus
PubMed: 18276775
DOI: 10.1124/mol.107.042028